Mortality from lung cancer could be reduced through implementation of chemopreventive strategies to reverse or impede progression of premalignant disease. Ingestion of L-selenomethionine has been associated with a 50% decrease in expected rates of lung cancer. ECOG 5597, "A Phase III Chemoprevention Trial of Selenium Supplementation in Persons with Resected Stage I Non-Small Cell Lung Cancer (NSCLC)," will test the hypothesis that selenium can decrease the rate of second primary tumors in patients who have undergone surgery for stage I NSCLC. Selenium may prevent lung cancer by protecting tissue from oxidative damage, inducing apoptosis of premalignant cells, and/or by acting as a demethylating agent through inhibition of cytosine DNA-methyltransferase. The identification of biomarkers that predict the efficacy of selenium would aid in the validation of this agent as a chemopreventive. We have targeted genes inactivated by CpG island methylation as candidate biomarkers and demonstrated that aberrant methylation of the p16 and O[6]-methylguanine-DNA-methyltransferase (MGMT) promoters can be detected in DNA from sputum in 100% of patients with squamous cell carcinoma up to 3 y before clinical diagnosis. Moreover, the prevalence of these markers in sputum from cancer-free, high-risk subjects approximates lifetime risk for lung cancer. Additional studies indicate that adenocarcinoma can also be detected through analysis of these and other genes in sputum and/or plasma. We have now extended these studies to begin developing a panel of methylation markers for predicting the development of lung cancer. In this study of sputum samples from 25 incident lung cancer cases and 25 matched controls, the presence of any of the four methylation markers examined was associated with a 6.3-fold increase in the risk for lung cancer. Thus, the use of aberrant gene methylation as a molecular marker system represents a powerful approach to evaluate the efficacy of selenium intervention. Using longitudinal collected sputum and plasma from subjects enrolled on ECOG 5597, we will first determine the prevalence for methylation of p16, MGMT, and death associated protein kinase after tumor resection. People positive for the methylation markers will be followed to determine how the methylation profile changes during intake of selenium versus placebo. Second, a nested, case-control study will determine whether changes in promoter hypermethylation predict development of a second primary lung cancer. These laboratory studies will specifically address mechanisms by which selenium is proposed to exert its chemopreventive effect. Our findings could identify molecular markers to monitor the efficacy of selenium supplementation in future prevention trials in cancer-free subjects with a history of smoking.